Macrophage Phagocytosis and MotilityMacrophages play important roles in host defense against invading micro-organisms and they are also key players in initiating and maintaining an immune response. However, macrophages can also play negative roles, such as in chronic inflammatory disease. Also, tumor-associated macrophages (TAMs), which are present in large numbers in many tumors, appear to play an important role in promoting the progression of solid tumors to an invasive, metastatic phenotype. Macrophages are therefore a prime target for therapies, but it is important to elucidate the mechanisms by which they are recruited to and activated in tissues.

Studying the molecular mechanisms of phagocytosis
Among their many roles, macrophages are best known for their striking ability to engulf a large number of big (>0.5µm) particles that are very diverse in size and shape in a process called phagocytosis. Phagocytosis is important in many situations such as the clearance of pathogen and particles (bacteria, yeast, pollen and pollutants). Crucially, phagocytosis may also be a major player of cancer immunity by mediating the engulfment and killing of cancer cells. Phagocytosis requires actin assembly, pseudopod extension, and phagosome closure (Figure 1). Actin polymerization in response to particle binding requires the activation of members of the Rho GTPases, either Rac or Cdc42 for Fc gamma Receptor-mediated phagocytosis or Rho in the case of CR3-mediated phagocytosis. Both Rac and Cdc42 regulate the cytoskeleton in part through the activation of the Wiskott Aldrich Syndrome/ WASP verprolin-homologous (WASP/WAVE) family of proteins. RhoG, another family member, is also important for phagocytosis but the precise role of RhoG is currently unknown. We are exploring the roles of these signaling pathways as well as those regulating the myosin family of molecular motors in phagocytosis. We are employing a novel technique called traction force microscopy (Figure 2) to understand the roles of these factors in the protrusive forces needed to engulf the diverse particles of various sizes and stiffnesses found in nature.

Several human pathogens, such as Cryptococcus neoformans and Legionella pneumophila, are internalized by macrophages, escape destruction and grow and divide inside the macrophage. The phagocytic processes by which these organisms are internalized are currently unknown. We are dissecting the downstream signaling pathways that mediate these processes during phagocytosis.

Studying the molecular mechanisms of chemotaxis
The directed movement of cells in response to chemoattractants involves several complex, interrelated processes, including directional or chemotactic sensing, polarity, and motility. These processes are mediated by complex, interacting signaling pathways that appear to have many similarities but yet have distinct characteristics depending on the chemoattractant and receptor. Many of the signaling cascades utilized for phagocytosis are also required for chemotaxis yet they result in the appearance of different structures (Figure 3 and our review [Rougerie et al., Immunol. Rev. 2013]). We are currently dissecting the signaling pathways required for macrophage chemotaxis towards:
1. CSF-1, a growth factor for macrophage survival and differentiation produced by many tumors and found in high concentrations in arthritic joints;
2. Chemokines that direct monocyte recruitment to different tissues.

Determining the role of podosomes during monocyte/macrophage diapedesis
Recruitment of immune cells from the blood to sites of tissue damage and infection is crucial. However, prolonged inflammation could have deleterious effects leading to serious chronic inflammatory diseases such as ischemic reperfusion and rheumatoid arthritis. Therefore understanding the mechanisms by which monocytes/macrophages contribute to this process would have a significant impact on identifying pharmacological targets and strategies. Post injury inflammation features the release of pro-inflammatory mediators that activate circulating monocytes as well as the vascular endothelium. We are interested in deciphering the mechanism of this multistep monocyte-endothelium interaction known as diapedesis (Figure 4). This involves the ability of cells to adhere to vascular endothelium, migrate across the endothelial barrier followed by degradation of sub-endothelial matrix. Monocyte-derived cells form characteristic F-actin rich structures known as podosomes, which are known to be required for cell adhesion, motility and degradation through multiple barriers. We are currently investigating the different regulators of podosomes and how they regulate the different roles that podosomes could potentially play during diapedesis. As key regulators of the actin cytoskeleton, our lab and others have shown that Cdc42 and downstream effectors WASP and N-WASP are essential regulators of podosome formation and/or function. Using different 2D and 3D in vitro models, we are currently investigating the role of podosomes during the different steps of diapedesis. In addition, using live imaging we are interested in monitoring the dynamics of podosomes during this process. Interestingly, using our newly developed FRET-based biosensor for Cdc42 (Hanna and Miskolci et al. PLoS One 2014), we will be able to monitor the function of Cdc42 during diapedesis in both space and time.

Determining the role of macrophages in the tumor microenvironment It is now increasingly recognized that the tissue microenvironment plays a critical role in tumor progression, but most studies on tumor metastasis involve the use of endpoint assays or in vitro studies on cell lines. It appears that macrophages and tumor cells participate in a paracrine interaction, with the tumor cells secreting CSF-1 and macrophages secreting EGF, but the precise roles of this paracrine interaction in tumor metastasis are unknown. We have developed a number of in vitro assays that reconstitute paracrine interaction between macrophage and carcinoma cells that mimic in vivo interactions of macrophages and tumor cells in the tumor microenvironment that have been observed by intravital imaging using multiphoton microscopy (Dovas et al.,J. Microscopy 2012). We are currently using these assays to understand the roles of both soluble factors and direct interaction between macrophages and tumor cells through tunneling nanotubes to mediate long distance signaling to promote tumor metastasis.

Development of tools for live cell imaging
In order to determine the role of individual molecules in macrophage functions it is essential to understanding the timing and localization of specific protein involved. We have been actively involved in the generation of new probes for single live cell imaging including photoconvertible actin probes that label various structures in live cells (Figure 6A,). We have also developed and employed biosensors to monitor localized protein activity in live cells (Figure 6B, Cammer et al., J. Biol. Chem. 2009), including the recent work in collaboration with Dr. Louis Hodgson on the development of isoform specific single chain RhoGTPase biosensors (Hanna and Miskolci et al., PloS One 2014).

Rougerie, P., Miskolci, V. and Cox, D. (2013) Generation of membrane structures during phagocytosis and chemotaxis of macrophages: role and regulation of the actin cytoskeleton. Immunol. Reviews. 256:222-239.